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Chapter 29:

analytical chemistry
Learning outcomes
You should be able to:
explain and use the terms Rf value in thin-layer predict the number of peaks in a carbon-13 NMR
chromatography and retention time in gas spectrum for a given molecule
liquid chromatography, and interpret gasliquid analyse and interpret a proton NMR spectrum of a
chromatograms to find the percentage composition simple molecule to deduce the dierent types of
of a mixture proton present, the relative numbers of each type
use a mass spectrum to deduce the molecular of proton present, the number of non-equivalent
mass of an organic molecule, the number of carbon protons adjacent to a given proton and the possible
atoms in a compound using the M + 1 peak, and structures for the molecule
the presence of bromine and chlorine atoms in a predict the chemical shifts and splitting patterns of
compound using the M + 2 peak the protons in a given molecule
suggest the identity of molecules formed by simple in obtaining an NMR spectrum, describe the use of
fragmentation in a given mass spectrum tetramethylsilane, TMS, as the standard for chemical
analyse a carbon-13 NMR spectrum of a simple shift measurements, and the need for deuterated
molecule to deduce the dierent environments of the solvents, e.g. CDCl3
carbon atoms present and the possible structures for describe the identification of O H and N H protons
the molecule by proton exchange using D2O.
Cambridge International A Level Chemistry

Introduction
We can detect and identify any chemical
substance using a range of different techniques.
Specialised instruments have been developed
to carry out tests, such as gas-liquid
chromatography, nuclear magnetic resonance
(NMR) spectroscopy and mass spectrometry. The
instruments used are often very sensitive, so
chemical substances can be detected at very
low concentrations.

Figure 29.1 Instrumental analysis can quickly detect tiny


traces of banned substances in a urine sample taken from
an athlete during random testing. These checks are used to
monitor many sportspeople around the world and ensure that
competition is fair on the sportsfield.

Chromatography The Rf values (retardation factors) of substances are


calculated as shown in Figure29.3. The conditions must be
Paper chromatography identical to those quoted in the Rf data table, e.g. the same
You will be familiar with the technique of paper temperature and the same solvent used.
434
chromatography. It is used to separate mixtures as a Coloured substances can be seen directly on the
solvent moves up a piece of absorbent paper. We call the paper but others are sprayed with a chemical that
solvent the mobile phase, and water trapped between the forms coloured compounds on the chromatogram. For
cellulose fibres of the paper is the stationary phase. The example, amino acids can be revealed as bluish spots by
substances in the mixture will have different affinities for ninhydrin spray.
the solvent and for the water, and so they move at different
rates over the paper (Figure29.2).

a b

solvent front
supporting rod

paper clip
paper

glass tank
solvent

sample to be
separated
pure reference pure reference mixture of solutes
compounds compounds to be identified

Figure 29.2 a Paper chromatography. b The chromatogram produced. Components of the mixture can be identified by
comparison with pure reference compounds or by calculating Rf values (see Figure29.3) and comparing these values with those in
tables of data.
Chapter 29: Analytical chemistry

solvent front Partition coefficients in chromatography


The principle of partition of a solute between two solvents
(see chapter 7) helps us to understand more fully how the
components in a mixture are separated in chromatography.
y Rf value is x
y In paper chromatography the different partition
x coefficients of the components in a mixture correspond to
their relative solubilities in the two solvents. The mobile
phase is the solvent chosen. The other solvent is the water
trapped in the papers structure, which is the stationary
compound started here
phase. Figure29.5 shows solute molecules partitioned
between the mobile phase and a stationary liquid phase on
Figure 29.3 How to calculate Rf values, which are a solid support.
then compared with reference values obtained under The solutes in the mixture being separated are
identical conditions.
partitioned to different extents between the solvents
in the mobile and stationary phases. The greater the relative
Two-way chromatography solubility in the mobile phase, the faster the
Sometimes two or more components in a mixture can rate of movement as the mobile phase passes over the
have similar Rf values in a particular solvent. This means stationary phase.
that their spots on the paper chromatogram will overlap
solute molecules
and separation will be poor. This can happen when we dissolved in the stationary
hydrolyse a protein and try to identify the amino acid stationary liquid phase liquid phase
residues present. This is when two-way chromatography is
useful. In this technique, paper chromatography is carried
out as normal but then the chromatogram produced 435
is rotated by 90 and re-run in a different solvent. It is
unlikely that the Rf values will coincide in two different
solvents, so separation takes place (Figure29.4). solid support
Figure 29.5 Partition chromatography. The mobile phase
moves over the stationary liquid phase, carrying solute
Making a two-way paper chromatogram
particles with it. The filter paper is the solid support in paper
chromatography.
1st solvent front
tn2nd
orf solvent
tnevlos front
dn2
two solutes
que io
solvent front
1st solvent front

overlapping
1 Look at this
paper chromatogram:

rotate paper
through 90
mixture of original three solutes now
three solutes mixture completely separated

A B C
Figure 29.4 Two-way paper chromatography to separate
solutes with similar Rf values in a solvent. The technique
can also be used with thin-layer chromatography (see
page 436). a The solvent used was ethanol. Which sample of
ink, A, B or C, has the greatest relative solubility
in ethanol?
b Work out the Rf value of the ink whose partition
coefficient in ethanol and water lies between the
values of the other two inks.
Cambridge International A Level Chemistry

Thin-layer chromatography Note that although TLC is normally described as


In thin-layer chromatography, referred to as TLC, the adsorption chromatography, some partitioning does
stationary phase is a solid that adsorbs solute molecules occur if water is present. Both dried alumina and silica
onto its surface (Figure29.6). can become rehydrated. When this happens, water also
acts as a partitioning stationary phase together with the
solute molecules adsorbed on the adsorbing stationary solid phase.
surface of the stationary phase
TLC is quicker than paper chromatography and
can be used on smaller samples, making it useful in
forensic science, where it can be used to identify drugs
and explosive residues. For example, TLC is used for
polar solid surface the analysis of a substance that is suspected to be
Figure 29.6 Adsorption chromatography. The mobile phase cannabis. The stationary phase is silica sprayed with
moves over the stationary solid phase. silver nitrate solution, which is then dried. The mobile
phase is methylbenzene.
The solid stationary phase is usually alumina (Al2O3) or
silica (SiO2), which is made into a slurry with water and
que io
spread onto a microscope slide. This is then put into an
oven, where it dries out into a solid white coating on the 2 a TLC can separate mixtures of components. What
glass. A chromatogram is then made in a similar way to do we call the mechanism of separation usually at
paper chromatography (Figure29.7). work in TLC?
b A mixture of propanone and hexane was separated
on a TLC chromatogram using alumina as the
Making a thin-layer chromatogram stationary phase and methylbenzene as the
solvent. Which substance would you expect to rise
436
further up the chromatogram? Explain why.
lid

thin layer of
SiO2 or Al2O3
coated onto a High-performance liquid
glass or plastic chromatography
surface
High-performance liquid chromatography, referred to
as HPLC, uses partitioning to separate and identify the
components in a mixture. The stationary phase is a non-
volatile liquid, such as a long-chain hydrocarbon liquid,
mixture of bonded onto a solid support, e.g. small particles of silica.
solutes
This is packed tightly into a column. The solvent chosen
solvent for the mobile phase is usually polar, e.g. a methanol/water
solvent. This has to be forced under pressure through
the densely packed column where separation occurs
Figure 29.7 Thin-layer chromatography. (Figure29.8).
The tiny solid particles in the column have a very large
surface area over which partitioning can occur, resulting
Polar molecules have a greater attraction for a polar solid in excellent separation. The more polar components in
used as the stationary phase, and they are adsorbed more the mixture have a greater relative solubility in the polar
strongly onto its surface. Therefore they travel more slowly solvent. Therefore they are carried through the column
up the thin layer of alumina or silica, and separation faster than components whose molecules are more non-
occurs. Solutes are located on the chromatogram and polar (which dissolve better in the non-polar stationary
identified by comparing with standard known substances phase in the column). The detector records retention
or by calculating Rf values. times, i.e. how long it takes each component to pass
Chapter 29: Analytical chemistry

packed column
I ICI IBI IAI
separated materials
from the column
mobile
phase

detectors
solvent
delivery injector
system
waste collected

injection of sample recorder concentration


mixture containing A B C
A, B and C time
chart record

Figure 29.8 High-performance liquid chromatography.

through the column. The area under each peak recorded HPLC is used:
is proportional to the amount of solute emerging from the in medical research to separate peptides and proteins
column (Figure29.9). to analyse urine samples from athletes for banned
substances such as steroids or stimulants
for monitoring pollutants in the atmosphere and in rivers,
Recorder response


437
e.g. measuring levels of pesticides
by food standards agencies to check the accuracy of the
data on food labels.

Gasliquid chromatography
5 10 15 20 min Gasliquid chromatography, which is referred to as
Time / min GLC, is similar to HPLC but a gaseous sample enters the
Figure 29.9 The chromatogram from a vitamin E HPLC column. The column contains the stationary phase and
analysis carried out by a food scientist investigating the sample is moved through by an inert carrier gas. This
chilli peppers. method is used with gases, liquids and volatile solids (as
they must be in the form of a vapour). The apparatus is
shown in Figure29.10.

carrier gas
computer
(mobile
or recorder
phase) in
sample injected
through silicone
rubber septum

exit
injector detector Figure 29.10 Gasliquid
port
detector chromatography. The
injector
oven oven maintains a constant
oven
temperature, higher
column (15 m long, than the boiling point of
36 mm diameter) the components in the
column oven mixture to be analysed.
Cambridge International A Level Chemistry

As in all chromatography, the conditions must be For this method:


controlled in order to make comparisons with published the chromatogram must show peaks for all the components
databases. The chromatogram must be obtained using in the mixture
the same carrier gas, flow rate, stationary phase and all the components of the mixture must be separated
temperature that were used when the standard data was the detector must respond equally to the different
obtained. Figure29.11 shows a chromatogram obtained components so that peak area is directly proportional to
using GLC. the component concentration.

The amount of each component in a mixture is found by


expressing it as a percentage of the sum of the areas under
propanone

pentan-2-one
all the peaks. For example, for a mixture of three esters A,
ethoxyethane

B and C:
heptane
Recorder response

(approx.) % of ester A
hex-1-ene

peak area (or height) of A


ethanal

= ___________________________________
100

sum of the areas (or heights) of A, B and C

GLC is used in testing for steroids in competing athletes


and for testing the fuels used in Formula One motor
racing (Figure29.12). It is also used for medical diagnosis
in analysing blood samples. With GLC it is possible to
determine the percentages of dissolved oxygen, nitrogen,
0 5 10 15 20
time of carbon dioxide and carbon monoxide in blood samples
Time / min
injection as small as 1.0cm3. GLC is often combined with mass
spectrometry (see later in this chapter) to separate then
438 Figure 29.11 A gas chromatogram from a mixture of volatile
rapidly identify the components of a mixture.
organic compounds.

Analysis by gasliquid chromatography does have


some limitations. For example, similar compounds will
have similar retention times and if a newly discovered
compound is detected it will not have a match in the
computers database of retention times.

Determination of the percentage


composition of a mixture by GLC
For quantitative analysis, the component peaks are
first identified and then the area of each is measured.
The peaks are roughly triangular in shape so their area
is approximately:
_ 1 base height (i.e. the area of a triangle) Figure 29.12 GLC is used to check that the components in the
2 fuel used in Grand Prix cars conform to strict regulations.
The GLC machine usually measures the area of the
peak automatically and can print the results with the que io
chromatogram. If the peaks are very narrow or have
3 a For GLC separations explain:
similar base widths, then peak height may be used instead
i how retention time is measured
of peak area to estimate the proportion of components in
ii how the areas under the component peaks
a mixture.
are used.
b What can you use as an approximate measure of
the proportion of a component in a mixture from a
GLC chromatogram which produces sharp peaks?
Chapter 29: Analytical chemistry

Proton (1H) nuclear magnetic this small difference in energy


lies in the radiowave range
resonance
against
How NMR works

Energy
Nuclear magnetic resonance (NMR) spectroscopy is a
before field
widely used analytical technique for organic compounds. is applied with
NMR is based on the fact that the nucleus of each after field is applied
hydrogen atom in an organic molecule behaves like a tiny
magnet. The nucleus of a hydrogen atom consists of a Figure 29.14 Hydrogen (1H) nuclei will absorb energy in the
single proton. This proton can spin. The spinning motion radiowave range when they flip from the lower energy level,
of the positively charged proton causes a very small lining up with the applied magnetic field, to the higher energy
level, lining up against it.
magnetic field to be set up.
In NMR we put the sample to be analysed in a
magnetic field. The hydrogen nuclei (protons) either line molecular environments flip at different field strengths.
up with the field or, by spinning in the opposite direction, The different field strengths are measured relative to a
line up against it (Figure29.13). reference compound, which is given a value of zero. The
There is a tiny difference in energy between the standard compound chosen is tetramethylsilane (TMS).
oppositely spinning 1H nuclei. This difference corresponds TMS is an inert, volatile liquid that mixes well with
to the energy carried by waves in the radiowave range most organic compounds. Its formula is Si(CH3)4, so all
of the electromagnetic radiation spectrum. In NMR its H atoms are equivalent (i.e. they are all in the same
spectroscopy the nuclei flip between the two energy molecular environment). TMS only gives one, sharp
levels (Figure29.14). Only atoms whose mass number is an absorption, called a peak, and this peak is at a higher
odd number, e.g. 1H or 13C, absorb energy in the range of frequency than most other protons (Figure29.15). All
other absorptions are measured by their shift away from 439
frequencies that are analysed.
The size of the gap between the nuclear energy levels the TMS line on the NMR spectrum. This is called the
varies slightly, depending on the other atoms in the chemical shift (), and is measured in units of parts per
molecule (the molecular environment). Therefore, NMR million (ppm).
can be used to identify 1H atoms in different parts of
a molecule. This is easier to visualise by looking at an single peak
example. If we look at a molecule of methanol, CH3OH, we of TMS
can see that there are 1H atoms in two different molecular (which we use
as a standard)
environments. We have the 1H atoms in the CH3 group
and the 1H atom in the OH group. The energy absorbed
by the CH3 1H atoms is different from the energy
absorbed by the 1H atoms in OH. 0 chemical
shift ()
In NMR spectroscopy, we vary the magnetic field as
that is easier than varying the wavelength of radiowaves. Figure 29.15 The standard TMS peak used as a reference on
As the magnetic field is varied, the 1H nuclei in different NMR spectra.

this nucleus H direction of H this nucleus


Figure 29.13 Hydrogen magnetic field
is lined up with nucleus nucleus is lined up against
(1H) nuclei will line up applied in
the applied field the applied field
with or against an applied NMR spectrometer
magnetic field.
Cambridge International A Level Chemistry

que io Low-resolution NMR


A low-resolution NMR spectrum shows a single peak for
4 a Explain why tetramethylsilane (TMS) is used as a each non-equivalent hydrogen atom; an example is shown
standard in NMR spectroscopy. in Figure29.17.
b In NMR we use solvents such as Note how the zero point on the x-axis, chemical shift
tetrachloromethane to prepare samples for the (), is on the right of the spectrum and the shift increases
machine (Figure29.16).
in value going left.
There are three peaks on ethanols low-resolution
NMR spectrum. These correspond to the 1H atoms in
OH, CH and CH . Note how the heights of the
2 3
peaks vary. The area under each peak tells us the relative
number of equivalent 1H atoms responsible for that
particular chemical shift. The largest peak will be from
the CH3 hydrogen atoms, the middle peak from the
CH hydrogen atoms and the smallest peak from the
2
OH hydrogen. The relative areas under the peaks are
shown on the NMR spectrum by the labels 1H, 2H and 3H
(Figure29.17).
The type of H atom present can be checked against
tables of data (Table29.1) if you are using NMR to identify
unknown organic compounds.
Using Table29.1 we can see that the peak at about 1.2ppm
is caused by the CH3 hydrogen atoms (range 0.71.6ppm),
440 the peak at about 3.7ppm corresponds to CH2 hydrogen
atoms (range 3.34.3ppm) and the peak at about 5.4ppm is
due to the OH hydrogen atom.

3H
Absorption of energy

2H

1H

Figure 29.16 Samples dissolved in a solvent in narrow


tubes ready for NMR analysis.
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
i What is the molecular formula of / ppm
tetrachloromethane?
Figure 29.17 The low-resolution NMR spectrum of
ii Why do you think tetrachloromethane is used ethanol, CH3CH2OH.
as a solvent?
iii Solvents that contain deuterium, D, are also
used as solvents in NMR. Deuterium is the
isotope 2H. A substance in which 1H is
replaced by 2H is said to be deuterated. Why
would the deuterated solvent CDCl3 be used
instead of CHCl3?
Chapter 29: Analytical chemistry

Type of proton Chemical shift, / ppm


R CH3 0.71.6
N H OH
R 1.05.5(a)
R CH2 R 1.21.4
R3CH 1.62.0

O O O
RCH2 C 2.02.9
H3C C R2CH C

CH3 CH2R CHR2 2.32.7

N CH3 CH R
N 2
CHR
N 2 2.32.9
O CH 3
CH R
O 2
CHR
O 2 3.34.3
Br or Cl CH3 Br or Cl CH2R Br or Cl CHR2 3.04.2

OH 4.510.0(a)

CH CH 45.6.0
O O
C C 5.012.0(a)
NH2 NH
441

H 6.58.0

O
C 9.010
H
O
C 11.012.0(a)
H

Table 29.1 1H NMR chemical shifts relative to TMS. Chemical shifts are typical values that can vary slightly depending on the
solvent, concentration and substituents. (a)OH and NH chemical shifts are very variable (sometimes outside these limits and are
often broad. Signals are not usually seen as split peaks).

que io High-resolution NMR


As you can see in Table29.1, the chemical shifts are given over
5 Predict the number of peaks and the relative areas ranges, and the ranges for different types of hydrogen atoms
under each peak, where appropriate, on the low- do overlap. In some molecules where there is heavy shielding
resolution proton NMR spectrum of:
of the hydrogen nuclei by lots of electrons in surrounding
a methanol, CH3OH d propan-1-ol atoms, peaks are shifted beyond their usual range. In such
b benzene, C6H6 e propan-2-ol cases high-resolution NMR is useful. High-resolution NMR
c chloroethane, C2H5Cl f propanone. gives us more information to interpret. Peaks that appear
as one signal on a low-resolution NMR spectrum are often
revealed to be made up of a cluster of closely grouped peaks.
This is because the magnetic fields generated by spinning
nuclei interfere slightly with those of neighbouring nuclei.
Cambridge International A Level Chemistry

This interference is called spinspin coupling. The exact Table29.2 shows the relative intensities and distribution of the
splitting pattern of a peak depends on the number of splitting patterns you are likely to meet.
hydrogen atoms on the adjacent carbon atom or atoms. Figure29.19 shows another high-resolution NMR
spectrum. You should try to interpret it by following
The number of signals a peak splits into equals n + 1 these steps:
where n is the number of 1H atoms on the adjacent Step 1 Use values to identify the environment of the
carbon atom.
equivalent protons (1H atoms) present at each
peak (remembering the peak at zero is the TMS
The high-resolution NMR spectrum of ethanol illustrates this standard reference peak).
n + 1 rule used to interpret splitting patterns (Figure29.18). Step 2 Look at the relative areas under each peak
The CH3 peak is split into three because there are two 1H to determine how many of each type of non-
atoms on the adjacent CH2 group. n + 1 = 3 (as n= 2); this is equivalent protons (1H atoms) are present.
called a triplet.
Step 3 Apply the n + 1 rule to the splitting patterns to see
The CH2 peak is split into four because there are three
1H atoms on the adjacent CH group. n + 1 = 4 (as n = 3);
which protons (1H atoms) are on adjacent carbon
3 atoms in the unknown molecule.
this is called a quartet.
The OH peak is not usually split as its 1H atom is Step 4 Put all this information together to identify the
constantly being exchanged with the 1H atoms of other unknown molecule.
ethanol molecules and any water present. This results in
one average peak being produced.

3H
1H
Absorption of energy

442
2H

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
/ ppm

Figure 29.18 The high-resolution NMR spectrum of ethanol, showing the splitting pattern in two of the peaks. The area under
each series of peaks still represents the number of equivalent 1H atoms in the molecule, as in low-resolution NMR.

Number of Using the n + 1 rule, the Relative intensities in the Observed on the NMR spectrum as
1
adjacent H atoms peak will be split into splitting pattern

0 1 peak, called a singlet 1

1 2 peaks, called a doublet 1:1

2 3 peaks, called a triplet 1:2:1

3 4 peaks, called a quartet 1:3:3:1

Table 29.2 Splitting patterns in high-resolution NMR spectra.


Chapter 29: Analytical chemistry

o ked exa ple que io

6 A pathologist was given a sample of a white tablet


1 An ester is used as a solvent in a glue. A chemist was
found at the scene of a suicide. In order to complete
given a sample of the ester to analyse. The NMR
her report the pathologist received an NMR spectrum
spectrum of the ester is shown in Figure29.19.
of the sample (Figure29.20a) and information from the
3H police that the tablets involved were either aspirin or
paracetamol. The displayed formulae of both drugs
Absorption of energy

are also shown in Figure29.20b.


3H a 3H

2H

Absorption of energy
6 5 4 3 2 1 0 1H
Chemical shift, / ppm
2H 2H
Figure 29.19 The high-resolution NMR spectrum of
an unknown ester in a glue. 1H

Step 1 Identify possibilities for the three major


peaks that appear at chemical shifts of 1.3, 11 10 9 8 7 6 5 4 3 2 1 0
1.9 and 4.1ppm. Using Table29.1, these Chemical shift, / ppm
could be:
b H O
1.3ppm R CH3, R CH2 R H H
H C H
1.9ppm R3CH (possibly H3C CO , O H H
443
RCH CO or R CH CO )
2 2 O N
H
H O
4.1ppm O CH , O CH R, O CHR C C H
3 2 2
H C H
H H O H
Step 2 Use the relative numbers of each type of O C
H H
proton (1H atom) labelling each major peak aspirin paracetamol
to narrow down possibilities.
Figure 29.20 a NMR analysis of the unknown drug
1.3ppm labelled 3H, so could be R CH3
sample. b Aspirin and paracetamol.
1.9ppm labelled 3H, so could be H C CO 3
4.1ppm labelled 2H, so could be O CH2R a Using this information, which drug was in the white
tablet? Explain your answer.
Step 3 By applying the n + 1 rule to the splitting
b Sketch the NMR spectrum you would expect to
patterns we can see which protons
see if the other drug had undergone NMR analysis.
(1H atoms) are on adjacent carbon atoms.
Label each peak with its relative area and the type
1.3ppm labelled 3H and split into triplet, so R CH3 of proton that caused it.
would be next to a C atom bonded to two
1H atoms (2 + 1 =3, triplet).

1.9ppm labelled 3H and a singlet, so H C CO 3


would be next to a C atom with no 1H atoms
attached (0 + 1 = 1, singlet). It could well be Identifying the OH or NH signal in an
next to the C O, with the carbonyl carbon NMR spectrum
also bonded to an O atom, as in an ester, i.e. The OH signal in the high-resolution NMR spectrum
H3C COOR.
of ethanol appears as a single peak. As we have seen
4.1ppm labelled 2H and split into quartet, O CH2R earlier, the peak is not split by the 1H atoms (protons)
would be next to a C atom bonded to three
1H atoms (3 + 1 = 4, quartet).
on the neighbouring CH2 group. The reason for this is
that the OH proton exchanges very rapidly with protons
Step 4 Putting this information together we get the in any traces of water (or acid) present, as follows:
ester ethyl ethanoate, CH3C CH2CH3.
CH3CH2OH + H2O CH3CH2OH + HOH
Cambridge International A Level Chemistry

The hydrogen atoms involved in this reversible proton


que io
exchange have been coloured red and blue. The exchange takes
place so rapidly that the signal for the OH protons becomes 7 a Look back to Figure29.18 on page 442. The high-
a single peak. This exchange also happens in amines and resolution NMR spectrum shown is from a sample
amides which contain the NH group. of ethanol containing traces of water. How would
Table29.3 shows the chemical shift ranges for the the NMR spectrum differ if D2O had been added to
different OH and NH signals. the sample of ethanol?
b Look back to uestion 6. How would repeating
the NMR analysis using a solvent of D2O be able to
Different OH and NH protons Range of chemical
help the pathologist distinguish between aspirin
shift () / ppm and paracetamol?
in alcohols, R OH protons 1.05.5

in phenols, arene OH protons 6.57.0

in carboxylic acids, R COOH protons 11.011.7 Carbon-13 NMR spectroscopy


In addition to proton NMR. carbon-13 NMR is another
in amines, NH2 / NH 1.05.5
analytical tool used frequently by organic chemists. The
in aryl amines, arene NH2 3.06.0 vast majority of carbon atoms in any organic compound
will be the carbon-12 isotope. This isotope has an even
in amides, CONH2 , CONH 5.012.0
mass number (12). Therefore it has no signal on an NMR
Table 29.3 Chemical shift ranges for OH and NH spectrum, as NMR only works with atoms with an odd
protons in different molecular environments. mass number (such as 1H, as we have already seen).
However, about 1% of the carbon atoms in any sample of
As you can see from Table29.1, these ranges overlap an organic compound will be the carbon-13 isotope. Its
444 with the chemical shifts of other types of proton. The nuclei will interact with the magnetic field applied in NMR
signals can also appear outside the quoted ranges analysis so can produce a NMR spectrum.
under certain conditions, e.g. choice of solvent or Carbon-13 NMR produces a spectrum with different
concentration. This makes NMR spectra difficult to chemical shifts for non-equivalent carbon atoms in a
interpret. However, there is a technique for positively molecule. Typical carbon-13 NMR shifts are shown in
identifying OH or NH groups in a molecule. Table 29.4. As in proton NMR, the chemical shifts are
Their peaks disappear from the spectra if you add a measured with reference to the TMS peak at 0 ppm on the
small amount of deuterium oxide, D2O, to the sample. spectrum.
The deuterium atoms (2H) in D2O, called heavy water, Analysis of carbon-13 NMR spectra is similar to that
exchange reversibly with the protons in the OH or of proton NMR, looking to match different chemical shifts
NH groups. For example: to characteristic molecular environments. However, the
signals produced in carbon-13 NMR appear as discrete
OH + D2O OD + HOD vertical lines on the spectra (without the complication of
NH CO + D2O ND CO + HOD the splitting patterns caused by the protons in 1H atoms
within the molecules). Take care in interpreting the
The deuterium atoms do not absorb in the same region carbon-13 NMR spectra because the heights of the lines
of the electromagnetic spectrum as protons (1H atoms). are not usually proportional to the number of equivalent
This makes the OH or NH signal disappear from the NMR 13C atoms present.

spectrum. By checking against the peaks in the original NMR The solvent used to prepare samples for 13C NMR
spectrum, without D2O, we can tell if the OH or NH analysis is CDCl3. This accounts for the small signal near
groups are present in the sample. The 1H atom in the OH or 80ppm that can be ignored when interpreting a spectrum,
NH group is referred to as a labile proton. as it is caused by the atoms of 13C in the solvent molecules.
Chapter 29: Analytical chemistry

Hybridisation of Environment of carbon atom Example structures Chemical / ppm


carbon atom shift range ()
sp3 alkyl CH3 , CH2 , CH 050
sp3 next to alkene/arene CH C C, CH 1040
2 2

sp3 next to carbonyl/carboxyl CH COR, CH CO R 2550


2 2 2
sp3 next to nitrogen CH NH , CH NR , CH NHCO 3065
2 2 2 2 2
sp3 next to chlorine ( CH2 Br and CH Cl
2 3060
CH I are in the same range
2
as alkyl)
sp3 next to oxygen CH OH, CH O CO 5070
2 2
sp2 alkene or arene C 110160
C C
C C ,
C C
C
sp2 carboxyl R CO2H, R CO2R 160185
sp2 carbonyl R CHO, R CO R 190220
sp2 alkyne R C C 6585
sp2 nitrile R C N 100125
Table 29.4 Typical 13C chemical shift values () relative to TMS = 0. Note that chemical shifts are typical values and can vary
slightly depending on the solvent, the concentration and substituents present. 445

Figure 29.21 shows the 13C NMR spectrum for propanone, Figure 29.22 shows another example of a carbon-13 NMR
(CH3)2CO. spectrum, that of ethylbenzene, C6H5CH2CH3.
Note that there are only two peaks: one for the carbon The carbon atoms in the benzene ring are almost
atom in the carbonyl group, C O, and the other for the equivalent but will be affected to slightly different extents
carbon atoms in the methyl groups, CH3. Although by the presence of the ethyl group in the molecule. Hence
there are two CH3 groups in propanone, they are both the series of lines clustered near 125ppm.
equivalent and so appear as only a single peak (just as
equivalent H atoms do in proton NMR).
C6H5CH2CH3
H3C
C O
H3C

200 100 0 200 100 0

Figure 29.21 The carbon-13 NMR spectrum of propanone. Figure 29.22 The carbon-13 NMR spectrum of ethylbenzene.
Cambridge International A Level Chemistry

The peak at the highest mass-to-charge ratio is caused by


que io
the molecular ion (M+). This ion is formed by the sample
8 a Look at the series of lines clustered at about molecule with one electron knocked out. It gives us the relative
125ppm in Figure 29.22. molecular mass of the sample. We can assume the
i One line is separated slightly from the main ions detected carry a single positive charge, so the reading
cluster explain which carbon atom in on the horizontal axis gives us the mass. In the case of
ethylbenzene is most likely to have produced propanone, CH3COCH3, the molecular ion has a relative
that signal. mass of 58.0. This corresponds to CH3COCH3+, with a mass
ii Predict how many lines make up the tightly of (3 12.0) + (1 16.0) + (6 1.0).
clustered grouping of the tallest line on the 13C We also get large peaks at 15 and 43 on the mass
spectrum and explain your reasoning. spectrum. These peaks are due to fragments that are
b Predict the number and location of signal lines in produced when propanone molecules are broken apart
the carbon-13 NMR spectrum of benzene, C6H6. by the electron bombardment. Knowing the structure
c Explain the number of signal peaks you would of propanone we should be able to identify the fragment
expect to see in the carbon-13 NMR spectrum of:
responsible for each peak (Figure29.24).
i propan-1-ol
ii propan-2-ol.

Mass spectrometry
You have already seen how a mass spectrometer
works (see pageX). The mass spectrum of an element
can be used to measure relative isotopic masses and
446 Figure 29.24 The fragmentation of propanone: +CH3 causes
their relative abundances. This information is used to
the peak at 15 and CH3C+O causes the peak at 43.
calculate relative atomic masses. However, the main
use of mass spectrometry is in the identification of
The electron bombardment has caused the C C single
organic compounds. As in other forms of spectroscopy,
bonds in the propanone molecules to break. This has
a substance can be identified by matching its spectrum
resulted in the fragments at m/e 15 and 43 that are
against the spectra of known substances stored in a
observed in Figure29.22. The breaking of single bonds,
database. This technique is known as fingerprinting.
such as C C, C O or C N, is the most common cause
In a mass spectrometer the sample is first vaporised.
of fragmentation.
When vapour from the sample enters the machine it
is bombarded by high-energy electrons. This knocks
electrons from the molecules and breaks covalent bonds, que io
fragmenting the molecule. Figure29.23 shows the mass
spectrum produced by propanone. 9 Look at Figure29.25 on page 447, which shows the
mass spectrum of ethanol, C2H5OH. A structural
100 isomer of ethanol is methoxymethane, an ether with
43
the formula CH3OCH3.
Relative abundance (%)

80
a Predict the mass-to-charge ratio of a fragment
60 that would appear on the mass spectrum of
58 methoxymethane but does not appear on
40 ethanols mass spectrum.
b Give the formula of the ion responsible for the
20 15 peak in your answer to part a.
0
0 20 40 60 80 100
Mass-to-charge ratio (m/e)

Figure 29.23 The mass spectrum of propanone.


Chapter 29: Analytical chemistry

que io ( o i ued) [CH2OH]+


100
c Look at the mass spectrum of ethanoic acid:

Relative abundance (%)


80
100 43 60
Relative abundance (%)

80 45 [C2H5]+ [C2H5O]+
40
[C2H3]+ [C2H5OH]+
60 15 20
60 [M + 1]
40
0
20 10 20 30 40 50
Mass-to-charge ratio, m/e

20 40 60 80 100 Figure 29.25 The mass spectrum of ethanol, showing the


Mass-to-charge ratio (m/e) [M + 1] peak.

Identify the fragments with mass-to-charge In any organic compound there will be 1.10% carbon-13.
ratios of: We can use this fact to work out the number of carbon
i 15 iii 45 atoms (n) in a molecule. We apply the equation:
ii 43 iv 60.
abundance of [M + 1]+ ion
100 ______________________
n = ___
1.1 abundance of M+ ion

High-resolution mass spectra


High-resolution mass spectrometers can distinguish o ked exa ple
between ions that appear to have the same mass on a low-
resolution mass spectrum. Table29.5 shows the accurate 2 An unknown compound has a molecular ion peak, M+, 447
with a relative abundance of 54.5% and has an [M + 1]+
relative isotopic masses of the most common atoms found
peak with a relative abundance of 3.6%. How many
in organic molecules. carbon atoms does the unknown compound contain?
Substituting the values of relative abundance into
Isotope Relative isotopic mass
the equation:
1H
1.0078246
abundance of [M + 1]+ ion
100 ______________________
12C
12.0000000 (by definition) n = ____
1.1



abundance of M+ ion
14N
14.0030738 we get:
16O
15.9949141
100 ____ 3.6
n = ____
1.1 54.5= 6.0
Table 29.5 Accurate masses of isotopes.
There are 6 carbon atoms in each molecule.
These accurate isotopic masses enable us to measure
the mass of the molecular ion so accurately that it can
only correspond to one possible molecular formula. For
example, a molecular ion peak at 45 could be caused by que io
C2H7N or CH3NO. However, a high-resolution mass 10 A hydrocarbon has a molecular ion peak at a mass-to-
spectrum would show the C2H7N+ peak at 45.057846 and charge ratio of 84 (relative abundance of 62.0%) and
the CH3NO+ peak at 45.021462. We can, therefore, be sure an [M + 1] peak with a relative abundance of 4.1%.
which molecule is being analysed. a How many carbon atoms are in the hydrocarbon?
b What is its molecular formula?
Using the [M + 1] peak
c The hydrocarbon does not decolourise bromine
There will always be a very small peak just beyond the water. Name the hydrocarbon.
molecular ion peak at a mass of [M + 1]. This is caused
by molecules in which one of the carbon atoms is the 13C
isotope. This is shown in the mass spectrum of ethanol in
Figure29.25.
Cambridge International A Level Chemistry

Using [M + 2] and [M + 4] peaks 100


112
[C6H535Cl]+
If the sample compound contains chlorine or bromine
atoms, we also get peaks beyond the molecular ion peak 80
because of isotopes of chlorine and bromine. Chlorine

Relative abundance (%)


77
has two isotopes, 35Cl and 37Cl, as does bromine, 79Br and
81Br. Table29.6 shows the approximate percentage of each 60 [C6H5]+

isotope in naturally occurring samples.


40
114
Isotopes Approximate % 51
+ [C6H537Cl]+
35Cl 75 20 [C4H2]+ [C4H3]

37Cl 25
35 56
79Br 50 0
30 50 70 90 110
81Br 50 Mass-to-charge ratio, m/e
Table 29.6 Naturally occurring isotopes of chlorine Figure 29.26 The mass spectrum of chlorobenzene, showing
and bromine. the [M + 2] peak. (Note that there are also tiny [M + 1] and
[M + 3] peaks corresponding to 13C in the molecule.)
One Cl or Br atom per molecule
The M, [M + 2] and [M + 4] peaks also occur in
Imagine a sample of chloromethane, CH3Cl. We will have
dibromomethane but the relative heights of peaks are
molecules of CH335Cl (75%) and molecules of CH337Cl
easier to work out. Because the ratio 79Br:81Br is 1:1, the
(25%). The molecular ion will be CH335Cl+, and two units
M:[M + 2]:[M + 4] height ratio is 1:2:1.
beyond that on the mass spectrum will be the peak for
CH337Cl+. The peak for CH337Cl+ will be one-third the
448 height of the molecular ion. This is the [M+ 2] peak. que io
In the mass spectrum of bromomethane, CH3Br, we
will have two molecular ion peaks of approximately the 11 a List the ions responsible for the M, [M + 2] and
same height one for CH379Br+ and the other for CH381Br+ [M + 4] peaks in a mass spectrum of dibromomethane.
(the [M + 2] peak). b What would be the mass-to-charge ratio and
You should look out for the relative heights mentioned relative abundances of the major peaks with the
highest charge-to-mass ratios in the mass spectrum
here when interpreting mass spectra.
of chloroethane?
if the [M + 2] peak is one-third the height of the M peak, this c How many peaks would you see beyond the
suggests the presence of one chlorine atom per molecule molecular ion peak in 1,1-dibromoethane? What
if the [M + 2] peak is the same as the height of the would be their mass-to-charge ratios and
M peak, this suggests the presence of one bromine abundances relative to the molecular ion? (Ignore
atom per molecule. peaks due to 13C.)
An example of the [M + 2] peak is shown on the mass
spectrum of chlorobenzene (Figure29.26).

Two Cl or Br atoms per molecule Applications of the mass spectrometer


The situation is a little more complex with two chlorine To identify the components in a mixture, we can link
atoms in a molecule, as there are three possibilities. gasliquid chromatography (GLC) or high-performance
Considering dichloromethane, CH2Cl2, we have: liquid chromatography (HPLC) apparatus directly to a
mass spectrometer.
35ClCH 35Cl+ the M peak
2 This combined technique is very sensitive, and any two
35ClCH 37Cl+ the [M + 2] peak
2 solutes that can be separated with a time gap of 1 second
37ClCH 35Cl+ the [M + 2] peak
2 on a GLC column can be identified almost instantly by
37ClCH 37Cl+ the [M + 4] peak
2 the mass spectrometer without the need to be collected.
The relative heights of the peaks must take into account Identification is by comparing the mass spectrum of
the natural abundances: it works out as 9:6:1 for each solute with the mass spectra of known compounds,
molecules with two Cl atoms. using a computers spectral database. The data generated
Chapter 29: Analytical chemistry

is complex. There can be many components in a mixture,


que io
each with a peak at its particular retention time on the
chromatogram, and each peak will generate its own 12 Look at Figure29.27.
characteristic series of lines in the mass spectrometer. a What is the retention time of the
We can combine the chromatogram and the mass spectra compound shown?
to display the data on a three-dimensional (3-D) graph b What is the approximate relative molecular mass
(Figure29.27). of the compound shown?
GLC linked to a mass spectrometer (GLCMS) is c How would the compound be identified?
used for analysing complex mixtures, for example the
identification of the hydrocarbons in a sample of crude oil.
The combined technique is fast and gives reliable results As with electrophoresis and NMR spectroscopy, mass
that can identify trace quantities of pollutants, drugs, spectrometry is also helping in medical research to both
biochemical molecules and toxins. This means it is used in: identify and research the amino acid sequences in proteins.
forensics It can be used to analyse the whole protein molecule or the
environmental monitoring of pollutants peptides left after breaking down the protein with specific
drug testing in sport enzymes. Figure29.28 shows the mass spectrum of a
geological and archaeological dating pentapeptide that is made into a charged compound by the
airport security. addition of a proton, hence the MH+ peak.
In research laboratories synthesising new compounds,
Mass spectrometry has even been used on space probes to
a combination of instrumental techniques will need to be
analyse rocks on Mars and in 2005 a mass spectrometer
used to confirm the structure of a previously undiscovered
was used to analyse the frozen hydrocarbon surface of
molecule (as no spectral records exist in databases, so
Titan, one of Saturns moons. The technique is also used to
identification by fingerprinting is not an option).
analyse the isotopes in the solar wind on board the Solar
and Heliospheric Observatory (SOHO) satellite. 449

100 200
180
80
Relative abundance (%)

160
60 140
120
40
e

100
m/

80
20
60
0 40
8.8 8.9 9.0 9.1
Time / min

Figure 29.27 The x-axis shows retention time, the y-axis the amounts and the z-axis is the charge/mass ratio of the mass spectra.
These 3-D data show the peaks on a mass spectrum for one component in a gasliquid chromatogram.
Cambridge International A Level Chemistry

100
MH+ que io

13 Look at Figure29.28.
Abundance (%)

a Calculate the relative molecular mass of


50
leucine enkephalin (C28H37N5O7) using relative
atomic masses.
(Ar values C =12.0, H = 1.0, N = 14.0, O = 16.0)
381.1
0 b i How is the peptide ionised before detection in
150 200 250 300 350 400 450 500 550 600 650 the mass spectrometer?
m/e
ii Why is this known as soft ionisation?
Figure 29.28 The mass spectrum of leucine enkephalin c Why is there a peak at [MH + 1]?
(C28H37N5O7), a peptide made up of five amino acids. It has d An unexpected peak occurs at charge-to-mass
been charged by adding a proton instead of by ionisation by ratio 578.1. This is caused by ionisation of the
high-energy electrons (which would fragment the molecule). pentapeptide by a metal ion instead of an H+ ion.
This is known as a soft ionisation method. Which metal ion is responsible for this ionisation?

Summary
Chromatography separates mixtures of substances Protons in different chemical environments
for identification. In chromatography, the mobile produce signals at different chemical shifts. The
phase moves the components of a mixture through chemical shift provides information about the
450 or over the stationary phase. Separation occurs by protons environment.
the transfer of the components to the stationary Protons on neighbouring carbon atoms cause signals
phase either by: to be split. The splitting pattern establishes which
partition between two liquids (due to the groups of protons are on adjacent carbon atoms. The
different solubility of solutes in the mobile phase n + 1 rule predicts the splitting pattern.
and stationary phase) Protons on OH and NH can be identified
partition between a gas and a liquid by the addition of D2O to the NMR sample, which
adsorption on a solid surface. collapses the peak due to an OH or an NH
The stationary phase may be solid or liquid; the proton.
mobile phase may be liquid or gas. Carbon-13 NMR can also help to determine the
In paper and thin-layer chromatography (TLC) the structure of organic molecules.
components of a mixture are identified by their The mass spectrum of a compound enables
Rf values. the relative molecular mass of the compound
In gasliquid chromatography (GLC) and high- to be determined using the molecular ion peak.
performance liquid chromatography (HPLC), the The molecular ion peak, M, is the peak produced
components of a mixture are identified by their by the loss of one electron from a molecule of
retention times; the amount of each component is the compound.
found by measuring the area of each peak (estimates We can deduce the number of carbon atoms in a
can be made from peak heights). compound using the [M + 1] peak and the presence
The proton NMR spectrum of a compound provides of a single bromine or chlorine atom using the
detailed information about the structure of the [M + 2] peak (and two Cl or Br atoms by the [M + 4]
compound. In particular, the spectrum for the peak as well).
protons, 1H, in a compound can provide a complete We can also use mass spectroscopy to identify
determination of the compounds structure. unknown organic compounds by fingerprinting
Chapter 29: Analytical chemistry

(matching the spectrum to other known spectra). times but can be fingerprinted by their unique
The fragmentation peaks give us clues as to the mass spectra). It is used in airport security checks,
structure of the original molecule. food industries and in forensic, environmental and
Gasliquid chromatography/mass spectrometry medical testing.
(GLCMS) provides a more powerful tool for A combination of techniques (such as infra-red, NMR
identifying the components in a mixture than GLC and mass spectroscopy) must be used to confirm the
alone (compounds can have similar retention structure of newly discovered compounds.

End-of-chapter questions
1 a Identify the fragments that would cause peaks in the mass spectrum of HOCH2COCH3 with the following
m/e values:
i m/e = 15 [1]
ii m/e = 17 [1]
iii m/e = 31 [1]
iv m/e = 43 [1]
v m/e = 57 [1]
vi m/e = 59 [1]
b At what value for m/e would you find the molecular ion peak? [1]
Total = 7
2 The gasliquid chromatogram for a mixture of organic compounds is shown below.
90
pentane
80
70
60 octane
pentan-1-ol
50
40
A
30
20
10 B
0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
C
a Give the correct labels for a, B and C. [3]
b What percentage of the mixture is pentan-1-ol? [6]
c Give an explanation for the different retention times. [3]
d i How would the chromatogram change if the liquid in the stationary phase was much more polar? [1]
ii Explain your answer. [2]
e Why is gasliquid chromatography useful in testing for anabolic steroids in the blood of athletes? [2]
f Explain why the use of gasliquid chromatography linked to a mass spectrometer is so useful. [2]
g Why is it difficult to separate dyes using gasliquid chromatography? [2]
Total = 21